Spermidine treatment of MJD zebrafish resulted in autophagy induction and inhibition of the autophagy pathway prevented the improved swimming produced by spermidine treatment. (A) Cohorts of MJD zebrafish larvae were euthanised and processed for immunoblot analysis for ataxin-3, LC3B and GAPDH loading control. (B) Inhibition of the autophagy pathway with chloroquine resulted in a significant increase in full length ataxin-3 levels (p < 0.0459, n = 11–12). (C) Comparison of LC3II levels in lysates from zebrafish treated with chloroquine, versus those co-treated with chloroquine and spermidine revealed that spermidine treated produced a higher level of LC3II, indicative of induction of autophagy (p < 0.0167, n = 11–12). (D) Example swimming trajectories of MJD zebrafish larvae treated with vehicle, spermidine, chloroquine or spermidine + chloroquine (Sp + Chlor) during an escape response to darkness test showed that spermidine treated larvae spent more time swimming at fast speeds and less at slow speeds. Co-treatment with spermidine + chloroquine returned the swimming to more similar to the vehicle treated group. (E) Measurement of distances swum by the MJD zebrafish show that spermidine treatment significantly increased the distances swum but chloroquine co-treatment prevented the improvement produced by the spermidine treatment (p < 0.0295, n = 4–5). A two-way ANOVA was utilised for statistical analysis followed by Tukey post hoc. Data represents mean ± SEM. Data points reflected are experimental treatments of approximately 20–25 larvae
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